PRIOR RELATED APPLICATION

This application claims the benefit of and priority to U.S. Provisional Application No. 63/367,741, filed on July 6, 2022, which is hereby incorporated by reference in its entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS XML VIA EFS-WEB

The instant application contains a Sequence Listing which has been filed electronically in .xml format and is hereby incorporated by reference in its entirety. Said .xml copy, created on June 28, 2023, is named 0G2440-1391116. xml and is 14 kilobytes in size. It is hereby stated that the information recorded on the computer readable form is identical to the written sequence listing and does not include matter which goes beyond the disclosure in the international application as filed.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under Grant No. CA241007 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Chronic inflammation is a central etiologic factor in many diseases. Inflammation results in the recruitment of immune cells and activation of inflammatory cytokines. Specialized cellular proteins respond to wound healing in an attempt to repair and restore normal tissue; however, often the response is dysfunctional and fibrosis results. Fibrosis results from the activation of fibroblasts that proliferate, migrate, and produce extracellular matrix components, such as type I collagen, and express cytokines and chemokines. Matrix metalloproteases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) are the main regulators of extracellular matrix turnover in tissue fibrosis including hepatic fibrosis.

One receptor that mediates the activation of fibrosis with inflammation is the G- protein coupled receptor (GPCR) - the cholecystokinin (CCK) receptor. CCK receptors are over-expressed in gastrointestinal cancers and stimulate growth when activated by ligands CCK or gastrin. Stimulated CCK receptors, e.g., on tissue fibroblasts and pancreatic stellate cells, cause the cells to produce collagen to form the extracellular matrix in chronic pancreatitis and pancreatic cancer. CCK-B receptors are not normally found in the liver or pancreas. But, with inflammation from pancreatitis, pancreatic cancer, hepatitis, or nonalcoholic steatohepatitis (NASH), the CCK-B receptor becomes activated. Over time, chronic inflammation in the liver can lead to scarring and more serious complications such as cirrhosis or liver cancer. Most hepatocellular carcinoma develops in livers with stage 4 fibrosis or cirrhosis. Other cancers are also associated with fibrosis including pancreatic, lung, and breast cancers. . As fibrosis in the tumor microenvironment can reduce penetration of therapeutics agents, strategies to decrease fibrosis in tissues and cancers to improve therapy delivery are being studied. However, in order to determine whether these treatments are effective or to determine the stage of disease, a biopsy is typically required. The FDA currently requires liver biopsies to evaluate efficacy of agents for the treatment of NASH, and this requirement has deterred many from participating in clinical trials. Although liver biopsies are typically safe with minimal risk in healthy patients, subjects with cirrhosis may have an increased risk of bleeding or another serious complication from a liver biopsy. Therefore, non-invasive methods for diagnosing and monitoring liver disease are necessary.

SUMMARY

Provided herein are methods for diagnosing liver disease or risk of liver disease in a subject. The methods comprise (a) obtaining a first biological sample from the subject;

(b) measuring the level of one or more miRNAs selected from the group consisting of miR- 185- 5p, miR-378a-3p, miR708-5p, miR-346-5p, miR302d-3p, miR-103-3p, miR-142-5p, miR126, miR-711, miR135b-5p, miR-21a-5p, miR-27a-3p, miR30c-2-3p, miR-9-5p, and miR29b-3p in the sample to obtain an expression profile; and, (c) diagnosing the subject with liver disease or at risk of liver disease if the expression profile is an expression profile comparable to a profile of one or more subjects with liver disease or at risk of liver disease. Some methods further comprise measuring in the sample the level of one or more of metallopeptidase inhibitor 1 (TIMP1), alpha-2 macroglobulin, and hyaluronic acid.

Also provided are methods for treating liver disease in a subject. The methods comprise (a) diagnosing liver disease or risk of liver disease in the subject according to any of the methods described herein; (b) measuring the level of one or more miRNAs selected from the group consisting of miR-185- 5p, miR-378a-3p, miR708-5p, miR-346-5p, miR302d-3p, miR-103-3p, miR-142-5p, miR126, miR-711, miR135b-5p, miR-21a-5p, miR-27a-3p, miR30c-2-3p, miR-9-5p and miR29b-3p in a sample from the subject diagnosed with liver disease or risk of liver disease to obtain a pretreatment expression profile; (c) administering a first therapeutic agent to the subject diagnosed with liver disease or risk of liver disease; (d) measuring the level of one or more miRNAs selected from the group consisting of miR-185- 5p, miR-378a-3p, miR708-5p, miR-346-5p, miR302d-3p, miR-103-3p, miR-142-5p, miR126, miR-711, miR135b-5p, miR-21a-5p, miR-27a-3p, miR30c-2-3p, miR-9-5p and miR29b-3p in the sample to obtain a post-treatment expression profile; (e) comparing the pretreatment and post-treatment expression profiles; and (f) administering the first therapeutic agent if the posttreatment expression profile correlates with disease improvement; or (g) modifying the dosage regimen of the first therapeutic agent or administering a second therapeutic agent to the subject if the expression profile determined in step (d) is not significantly changed or correlates with disease progression. In some methods, the liver disease is fibrosis, hepatic inflammation, cirrhosis, or hepatocellular carcinoma.

In some methods, modifying the dosage regimen of the first therapeutic agent comprises increasing or decreasing the dosage of the first therapeutic agent. In some methods modifying the dosage regimen of the first therapeutic agent comprises administering the first therapeutic agent and the second therapeutic agent to the subject. In some methods, the second therapeutic agent is an immune checkpoint inhibitor or a chemotherapeutic agent. In some methods, the second therapeutic agent is an antiviral agent. In some methods, the first therapeutic agent is an anti-fibrotic agent. In some methods, the anti-fibrotic agent is proglumide.

In some methods, the disease is fibrosis, hepatic inflammation, pancreatitis, cirrhosis, or hepatocellular carcinoma.

Also provided are methods for treating nonalcoholic steatohepatitis (NASH) in a subject. The methods comprise (a) obtaining a first biological sample from the subject prior to administration of a first therapeutic agent (e.g., proglumide); (b) measuring the level of one or more miRNAs selected from the group consisting of miR-185- 5p, miR-378a-3p, miR708- 5p, miR-346-5p, and miR302d-3p in the first sample; (c) administering to the subject an effective amount of the first therapeutic agent; (d) obtaining a second biological sample from the subject following administration of the first therapeutic agent ; (e) measuring the level of one or more miRNAs selected from the group consisting of miR-185- 5p, miR-378a-3p, miR708-5p, miR-346-5p, and miR302d-3p in the second sample; (f) comparing the levels of the one or more miRNAs determined in steps (b) and (e); and (g) administering the first therapeutic agent to the subject subsequent to the comparing step (f) if the level of the one or more miRNAs determined in step (e) is higher than the level of the one or more miRNAs determined in step (b); or (h) modifying the dosage regimen of first therapeutic agent or administering a second therapeutic agent (e.g., a non-proglumide agent) to the subject subsequent to the comparing step (f) if the level of the one or more miRNAs determined in step (e) is not significantly changed or is lower than the level of the one or more miRNAs determined in step (b).

Other methods for treating NASH comprise (a) obtaining a first biological sample from the subject prior to treatment; (b) measuring the level of one or more miRNAs selected from the group consisting of miR-103-3p, miR-142-5p, miR126, miR-711, and miR135b-5p in the first sample; (c) administering to the subject an effective amount of a first therapeutic agent (e.g., proglumide); (d) obtaining a second biological sample from the subject following administration of the first therapeutic agent; (e) measuring the level of one or more miRNAs selected from the group consisting of miR-103-3p, miR-142-5p, miR126, miR-711, and miR135b-5p, in the second sample; (f) comparing the levels of the one or more mRNAs determined in steps (b) and (e); and (g) administering the first therapeutic agent to the subject subsequent to the comparing step (f) if the level of the one or more miRNAs determined in step (e) is lower than the level of the one or more miRNAs determined in step (b); or (h) modifying the dosage regimen of the first therapeutic agent or administering a second therapeutic agent (e.g., a non-proglumide agent) to the subject subsequent to the comparing step (f) if the level of the one or more miRNAs determined in step (e) is not significantly changed or is higher than the level of the one or more miRNAs determined in step (b).

In some methods, modifying the dosing regimen of a first therapeutic agent comprises increasing the dosage. In some methods, modifying the dosing regimen of the first therapeutic agent comprises administering the first therapeutic agent in combination with the second therapeutic agent to the subject.

Also provided are methods for treating hepatocellular carcinoma in a subject. The methods comprise (a) obtaining a first biological sample from the subject prior to treatment; (b) measuring the level of one or more miRNAs selected from the group consisting of miR- 21a-5p, miR-27a-3p, miR30c-2-3p, and miR-9-5p and miR29b-3p, in the first sample; (c) administering to the subject an effective amount of proglumide; (d) obtaining a second biological sample from the subject following treatment; (e) measuring the level of one or more miRNAs selected from the group consisting of miR-21a-5p, miR-27a-3p, miR30-2-3p, and miR-9-5p and miR29b-3p, in the second sample; (f) comparing the levels of the one or more mRNAs determined in steps (b) and (e); and (g) administering proglumide to the subject subsequent to the comparing step (f) if the level of the one or more miRNAs determined in step (e) is lower than the level of the one or more miRNAs determined in step (b); or (h) modifying the dosage regimen of proglumide or administering a non-proglumide agent to the subject subsequent to the comparing step (f) if the level of the one or more miRNAs determined in step (e) is not significantly changed or is higher than the level of the one or more miRNAs determined in step (b).

In some methods, modifying the dosage regimen of proglumide comprises increasing the dosage of proglumide. In some methods, altering the dosage regimen of proglumide comprises administering proglumide and a second therapeutic agent to the subject. In some methods, the second therapeutic agent is an immune checkpoint inhibitor or a chemotherapeutic agent.

In any of the methods provided herein, the sample can be a bodily fluid sample. In some methods, the bodily fluid is blood. In any of the methods described herein, the level of the one or more miRNAs is determined using hybridization, RT-PCR, or sequencing.

BRIEF DESCRIPTION OF THE DRAWINGS

The present application includes the following figures. The figures are intended to illustrate certain embodiments and/or features of the compositions and methods, and to supplement any description(s) of the compositions and methods. The figures do not limit the scope of the compositions and methods, unless the written description expressly indicates that such is the case.

FIGS. 1 A-B show proglumide blood levels per cohort measured by mass spectrometry. (A) Absolute and mean ± SEM values of blood proglumide levels shown for week 12 in the three separate cohorts. (B) Mean proglumide blood levels over time collected at baseline (week 0) and weeks 4, 8, and 12 for each cohort in the study.

FIG. 2 shows fold change in serum miRNA values according to treatment groups. Four miRNAs miR-185- 5p, miR-378a-3p, miR708-5p, and miR-346-5p) were measured at baseline and with proglumide therapy. All four miRNAs increased in serum of subjects treated with proglumide as measured by qPCR. Significant differences compared to baseline values using a two-sided Student’s T-test included: miRNA-378-3p, P=0.004; miRNA-708- 5p, PO.OOl, and miRNA-346-5p, PO.OOl.

FIG. 3 is a heat map of biomarkers from mice treated with vehicle (V) or proglumide (P). Those in a black box are anti-fibrosis miRNAs. Those in a red box are miRNAs that increase fibrosis and are down-regulated. Those in Green boxes are oncogenes associated with hepatocellular carcinoma (HCC).

FIG. 4 shows expression of miRNA markers associated with: fibrinolysis measured from circulating blood from mice bearing PDAC tumors. ** p < 0.05, *** p < 0.005 FIG. 5 shows expression of miRNA markers associated with EMT and cancer invasion measured from mice bearing PANC-1 orthotopic tumors. *p<0.05, **p < 0.01, *** p < 0.005

DETAILED DESCRIPTION

The following description recites various aspects and embodiments of the present compositions and methods. No particular embodiment is intended to define the scope of the compositions and methods. Rather, the embodiments merely provide non-limiting examples of various compositions and methods that are at least included within the scope of the disclosed compositions and methods. The description is to be read from the perspective of one of ordinary skill in the art; therefore, information well known to the skilled artisan is not necessarily included.

Diagnostic Methods

Provided herein is a method for diagnosing or staging liver disease or risk of liver disease in a subject comprising: (a) obtaining a first biological sample from the subject; (b) measuring the level of one or more miRNAs selected from the group consisting of miR-185- 5p, miR-378a-3p, miR708-5p, miR-346-5p, miR302d-3p, miR-103-3p, miR-142-5p, miR126, miR-711, miR135b-5p, miR-21a-5p, miR-27a-3p, miR30c-2-3p, miR-9-5p, and miR29b-3p in the sample to obtain an expression profile; and, (c) diagnosing the subject with liver disease or at risk of liver disease if the expression profile is an expression profile comparable to a profile of one or more subjects with liver disease or at risk of liver disease.

As used herein, liver disease, is defined as any condition that damages the liver and/or decreases liver function. Examples of liver disease include, but are not limited to, alcoholic fatty liver disease, liver fibrosis, nonalcoholic fatty liver disease (NASH), hepatitis C, hepatitis B, hepatitis A, pancreatitis, cirrhosis, hemochromatosis, and liver cancer (e.g., hepatocellular carcinoma (HCC).

As used herein, the term biological sample or sample refers to a sample that has been obtained from a patient or subject. In some instances, the biological sample can be a tissue, cell(s), a bodily fluid (e.g., blood, serum, synovial fluid, sputum, lung fluid, mucus, tears, lymphatic fluid, synovial fluid, cerebrospinal fluid, stool, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). A biological sample can also be a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or bodily fluids. In some instances, nucleic acids, for example, DNA or RNA is isolated from a tissue, cell or bodily fluid of the subject.

In some methods, the biological sample comprises blood from the subject. In some cases, the biological sample comprises plasma. In other methods, the biological sample may be obtained directly from a subject (e.g., by blood or tissue sampling) or from a third party (e.g., received from an intermediary, such as a healthcare provider or lab technician).

As used throughout, by subject is meant an individual. The subject can be an adult subject or a pediatric subject. Pediatric subjects include subjects ranging in age from birth to eighteen years of age. Preferably, the subject is an animal, for example, a mammal such as a primate, and, more preferably, a human. Non-human primates are subjects as well. The term subject includes cats, dogs, reptiles, amphibians, livestock (for example, cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.). Thus, veterinary uses and medical formulations are contemplated herein.

As used herein, the term diagnosis means detecting a disease or disorder or determining the stage or degree of a disease or disorder. The diagnostic methods may be used independently, or in combination with other diagnosing and/or staging methods known in the art for a particular disease or disorder. Additional diagnostic markers may be combined with the detection of miRNA level(s) to construct models for predicting the presence or absence or stage of a disease. For example, clinical factors of relevance to the diagnosis of liver disease include, but are not limited to, the patient's medical history, a physical examination, and other biomarkers. A diagnosis of liver disease may also be informed by a patient's symptoms, including the type of symptoms, duration of symptoms and degree of symptoms. For example, and not to be limiting, any of the methods described herein can be used in combination with measuring the level of one or more liver enzymes in a biological sample from the subject, imaging tests (for example, ultrasound, MRI, CT scan, etc.) and/or a liver biopsy. Some methods further comprise measuring hydroxyproline (e.g., by mass spectrometry (See, e.g., Jolly et al., Cancers 15:2811 (2023)), metallopeptidase inhibitor 1 (TIMP1), alpha-2 macroglobulin, and hyaluronic acid in a biological sample from the subject.

As used herein, microRNA or miRNA refers to small non-coding RNA molecules, generally about 15 to about 50 nucleotides in length, optionally between about 17-23 nucleotides, which can play a role in regulating gene expression through, for example, RNA interference (RNAi). RNAi describes a phenomenon whereby the presence of an RNA sequence that is complementary or antisense to a sequence in a target gene messenger RNA (mRNA) results in inhibition of expression of the target gene.

In any of the methods provided herein, the biological sample is assessed to determine the level of one or more miRNAs selected from the group consisting of miR-185- 5p, miR- 378a-3p, miR708-5p, miR-346-5p, miR302d-3p, miR-103-3p, miR-142-5p, miR126, miR-

711, miR135b-5p, miR-21a-5p, miR-27a-3p, miR30c-2-3p, miR-9-5p and miR29b-3p, associated with liver disease. In general, detecting a miRNA is carried out by determining the presence or absence of a nucleic acid comprising a miRNA of interest in the biological sample. Exemplary sequences that can be used to determine the level of one or more miRNAs described herein are set forth below in Table 1.

Table 1. Exemplary Sequences for miRNAs Nucleic acids, for example, RNA, including miRNA, can be obtained from the biological sample using known techniques. Nucleic acids can also be obtained from an extraction performed on a fresh or fixed biological sample.

Methods for determining the level of one or more miRNAs in a biological sample are known to those of skill in the art. For example, Northern analysis, in situ hybridizations, microarrays, bead-array profiling, RT-qPCR, amplification assays, next generation sequencing, or enzyme-based assay (e.g., Invader assay) can be used. For the quantification of active miRNA fluorescence or bioluminescence based optical imaging, magnetic resonance imaging, and positron emission tomography can be used to monitor activity.

Direct sequence analysis can also be used to detect miRNAs of interest. A sample comprising nucleic acid can be used, and PCR or other appropriate methods can be used to amplify one or more miRNA sequences in a biological sample.

Arrays of oligonucleotide probes that are complementary to target nucleic acid sequences from a subject can also be used to detect and quantify one or more miRNAs associated with liver disease. For example, in some embodiments, an oligonucleotide array can be used. Oligonucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. Provided herein is an array of oligonucleotide probes comprising one or more probes for detecting one or more miRNAs selected from the group consisting of miR-185- 5p, miR-378a-3p, miR708- 5p, miR-346-5p, miR302d-3p, miR-103-3p, miR-142-5p, miR126, miR-711, miR135b-5p, miR-21a-5p, miR-27a-3p, miR30c-2-3p, miR-9-5p, and miR29b-3p. miRNA can also be detected using nucleic acid amplification methods. Amplification refers to any method for increasing the number of copies of a nucleic acid sequence. For example, the amplification can be performed with a polymerase, e.g., in one or more polymerase chain reactions (PCR), using methods known in the art. Exemplary PCR methods include, but are not limited to, real time PCR (RT-PCR), droplet digital PCR, multiplex PCR, quantitative PCR etc. Stem-loop RT-PCR is a PCR method that is also useful in the methods described herein to amplify and quantify miRNAs of interest (See Caifu et al., 2005, Nucleic A cids Research 33 : e 179) .

The term expression profile, as used in the context of the present invention, represents the determination of a miRNA expression profile or a measure that correlates with the miRNA expression in a sample (e.g. in a blood sample). By determining the miRNA expression profile, each miRNA is represented by a numerical value. The higher the value of an individual miRNA, the higher is the expression level of said miRNA, or the lower the value of an individual miRNA, the lower is the expression level of said miRNA. Any of the expression profiles described herein can also include ratios of miRNAs in a sample. The expression profile may be generated by any convenient means, e.g. nucleic acid hybridization (e.g. to a microarray), nucleic acid amplification (PCR, RT-PCR, qRT-PCR, high-throughput RT-PCR), ELISA for quantitation, next generation sequencing (e.g. ABI SOLID, Illumina Genome Analyzer, Roche/454 GS FLX), flow cytometry (e.g. LUMINEX, Milipore Guava) and the like, that allow the analysis of an miRNA expression profile in a subject and comparison between samples. Expression profiling techniques are reviewed by Pritchard et. al (Nat Rev Genet. 2012, PMID:22510765) which is incorporated herein by reference in its entirety. A biological sample analyzed by any of the methods described herein can be a blood sample, and may include total RNA, labeled total RNA, amplified total RNA, cDNA, labeled cDNA, amplified cDNA, miRNA, labeled miRNA, amplified miRNA or any derivatives that may be generated from the aforementioned RNA/DNA species. As used herein, an expression profile comprises at least one miRNA, and optionally, at least two miRNAs, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more miRNAs, selected from the group consisting of miR-185- 5p, miR-378a-3p, miR708-5p, miR-346-5p, miR302d-3p, miR-103-3p, miR- 142-5p, miR126, miR-711, miR135b-5p, miR-21a-5p, miR-27a-3p, miR30c-2-3p, miR-9-5p and miR29b-3p.

The reference sample used in any of the methods described herein, for example, diagnostic or treatment methods, can be a sample derived from the same type of cell or tissue (e.g., blood) as a biological sample, and with a known condition. For example, the reference sample can represent a normal condition of a cell or tissue in a biological sample to be analyzed. Alternatively, the reference sample can represent a recognizable stage of an abnormal condition (e.g., liver disease) of a cell or a tissue (e.g., blood) in a biological sample to be analyzed.

In some methods, more than one reference sample can be used, wherein the reference samples represent a variety of different conditions (e.g., normal condition, different stages of a disease or disorder, etc.). By way of example, if a biological sample from a subject generates a similar or comparable miRNA expression profile (e.g., in terms of levels of miRNA sequences) to that of a normal sample or a group of normal samples (reference samples), the subject can be considered normal with respect to the normal sample or the group normal samples. Similarly, if a biological sample of a subject generates a similar or comparable miRNA expression profile (e.g., in terms of levels of miRNA sequences) to that of a sample or a group of disease samples, the subject's biological sample can be considered to have the same disease as the reference samples. In some methods, for example, treatment methods, if a biological sample of a subject generates a similar or comparable miRNA expression profile (e.g., in terms of levels of miRNA sequences) to that of a sample or a group of disease samples that correlates with disease progression, after treatment with a therapeutic agent, the subject can be categorized as not progressing, or not responding to the therapeutic agent. In other treatment methods, if a biological sample of a subject generates a similar or comparable miRNA expression profile (e.g., in terms of levels of miRNA sequences) to that of a sample or a group of disease samples that correlates with disease improvement, after treatment with a therapeutic agent, the subject can be categorized as responding to the therapeutic agent.

In some methods, cluster analysis can be computationally performed to classify miRNA expression profiles of a biological sample and a set of reference samples into groups such that samples in the same group (called a cluster) have more similar miRNA expression profiling to each other than to those in other groups (clusters). Accordingly, when a biological sample is categorized into the same group with a subset of reference samples of similar miRNA expression profiles, the biological sample is considered to have similar properties (e.g., genotype or phenotype) as the subset of reference samples. For example, when a biological sample from a subject is categorized into the same group representing liver cancer reference samples, or more specifically, into a sub-group representing HCC, the subject is determined to likely have liver cancer, or more specifically HCC. In another example, when a biological sample from a subject is categorized into the same group representing liver fibrosis reference samples, or more specifically, into a sub-group representing advanced liver fibrosis, the subject is determined to likely have liver fibrosis, or more specifically advanced liver fibrosis. Various clustering algorithms for classification and clustering are known in the art and can be used for the purposes described herein. Examples of clustering algorithms and/or models include, but are not limited to, connectivity-based clustering (e.g., hierarchical clustering), centroid-based clustering (e.g., k-means clustering), distribution-based clustering (e.g., multivariate normal distributions used by the expectationmaximization algorithm), density-based clustering (e.g., DBSCAN and OPTICS), subspace models (e.g., biclustering), and any combinations thereof. Any of these methods can be used in combination with principal component analysis, as described in the Examples.

The methods described herein can be used to assign treatment to a patient suffering from liver disease. By detecting an expression profile of the miRNA biomarkers described herein corresponding to a subject with liver disease or a stage of liver disease, the appropriate treatment can be assigned to a patient suffering from the disease. These treatments can include, but are not limited to, administration of a CCK receptor (CCK-A receptor, CCK-B receptor or CCK-C receptor) inhibitor, an antibiotic, an antiviral, chemotherapy, immunotherapy, radiation treatment, surgical treatment, FXR agonist, GLP-1 agonist, or any combination thereof.

For example, and not to be limiting, if a subject is diagnosed with NASH, the subject can be treated with a CCK receptor inhibitor, for example, proglumide. In another nonlimiting example, if a subject is diagnosed with HCC, the subject can be assigned to treatment with a chemotherapeutic agent and/or a CCK receptor inhibitor, for example, proglumide and/or gemcitabine. In yet another example, if the subject is diagnosed with Hepatitis B, the subject can be treated with an antiviral agent, for example, tenofovir, lamivudine, or adefovir, to name a few.

Treatment Methods

Also provided are methods for treating liver disease in a subject. The methods comprise (a) diagnosing liver disease or risk of liver disease according to any one of the methods described herein; (b) measuring the level of one or more miRNAs selected from the group consisting of miR-185- 5p, miR-378a-3p, miR708-5p, miR-346-5p, miR302d-3p, miR- 103-3p, miR-142-5p, miR126, miR-711, miR135b-5p, miR-21a-5p, miR-27a-3p, miR30c-2- 3p, miR-9-5p and miR29b-3p in a sample from the subject diagnosed with liver disease or risk of liver disease to obtain a pretreatment expression profile; (c) administering a first therapeutic agent to the subject diagnosed with liver disease or risk of liver disease; (d) measuring the level of one or more miRNAs selected from the group consisting of miR-185- 5p, miR-378a-3p, miR708-5p, miR-346-5p, miR302d-3p, miR-103-3p, miR-142-5p, miR126, miR-711, miR135b-5p, miR-21a-5p, miR-27a-3p, miR30c-2-3p, miR-9-5p and miR29b-3p in the sample to obtain a post-treatment expression profile; (e) comparing the pretreatment and post-treatment expression profiles; and (f) administering the first therapeutic agent if the posttreatment expression profile correlates with disease improvement; or (g) modifying the dosage regimen of the first therapeutic agent or administering a second therapeutic agent to the subject if the expression profile determined in step (d) is not significantly changed or correlates with disease progression. In some methods, the liver disease is fibrosis, hepatic inflammation, pancreatitis, cirrhosis, or hepatocellular carcinoma. In some methods, the first therapeutic agent is a CCK receptor inhibitor (e.g., proglumide), a chemotherapeutic agent, an antiviral, an immunotherapeutic agent or any other agent known or later identified to treat liver disease.

As used herein the terms treatment, treat, or treating refers to a method of reducing one or more of the effects of the disease or one or more symptoms of the disease, for example, liver disease, in the subject. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of liver disease. In addition to alleviation or prevention of symptoms, treatment can also slow or stop the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder or condition. For example, a method for treating NASH or HCC is considered to be a treatment if there is a 10% reduction in one or more symptoms of NASH or HCC in a subject as compared to a control. Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease or symptoms of the disease.

In some methods, modifying the dosage regimen of the first therapeutic agent comprises increasing or decreasing the dosage of the first therapeutic agent. In some methods modifying the dosage regimen of the first therapeutic agent comprises administering the first therapeutic agent and the second therapeutic agent to the subject. In some methods, the second therapeutic agent is vitamin E. In some methods, the second therapeutic agent is an immune checkpoint inhibitor or a chemotherapeutic agent. In some methods, the second therapeutic agent is an antiviral agent. In some methods, the first therapeutic agent is an anti- fibrotic agent. In some methods, the anti-fibrotic agent is a CCK receptor inhibitor.

In some methods, for example, treatment of liver cancer, a CCK receptor inhibitor (e.g., proglumide) and a chemotherapeutic agent (e.g., gemcitabine) are administered in combination as the first therapeutic treatment for the subject. In such cases, modification of the dosage of the CCK receptor inhibitor (e.g., proglumide) and/or the chemotherapeutic agent can be modified if the post-treatment expression profile does not correlate with disease improvement

In any of the methods provided herein, the CCK receptor inhibitor or antagonist can be a CCK receptor inhibitor or antagonist that inhibits one or more CCK receptors selected from the group consisting of a CCK-A (CCK-1) receptor, a CCK-B (CCK-2 or gastrin) receptor and a CCK-C receptor. In any of the methods provided herein, the CCK receptor antagonist can decrease fibrosis in the subject, for example, liver fibrosis. Optionally, administration of a CCK receptor antagonist decreases fibrosis, for example, NASH- associated fibrosis, in the subject. Optionally, administration of a CCK receptor antagonist decreases fibrosis associated with viral hepatitis (hepatitis B, hepatitis C and HIV fibrosis), alcoholic hepatitis, cirrhosis, autoimmune hepatitis, primary biliary cholangitis or sclerosing cholangitis.

In any of the methods provided herein, administration of a CCK receptor inhibitor can decrease inflammation in the subject with liver disease, for example, pancreatitis, NASH or HCC. In any of the methods provided herein, administration of a CCK receptor inhibitor can prevent the development or progression of cirrhosis in a subject having NASH and/or decrease fibrosis, for example, reverse established fibrosis in a subject. In any of the methods provided herein, administration of a CCK receptor inhibitor can prevent the development of progression of HCC in a subject having NASH. In any of the methods provided herein, a CCK receptor antagonist can also reverse established fibrosis of the liver in a subject.

In the methods described herein the chemotherapeutic agents that can be used include, but are not limited to, antineoplastic agents such as Acivicin; Aclarubicin; Acodazole Hydrochloride; AcrQnine; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflomithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized Oil 1 131; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; 5 -Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-nl; Interferon Alfa-n3; Interferon Beta- 1 a; Interferon Gamma- 1 b; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin C; Mitosper; Mitotane; Mitoxantrone; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;

Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safmgol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin;

Streptozocin; Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfm; Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride.

In any of the methods provided herein, where a chemotherapeutic agent is administered, the chemotherapeutic agent can be selected from the group consisting of paclitaxel, gemcitabine, fluorouracil and irinotecan.

In some methods, the CCK receptor inhibitor is administered in conjunction with a second therapeutic agent, such as a chemotherapeutic agent, to prevent development or progression of HCC. In some embodiments, the CCK receptor inhibitor and, optionally, a second therapeutic agent, are administered to a subject to prevent progression of pre-HCC to HCC. As used throughout, pre-HCC includes, but is not limited to, alcohol abuse, viral hepatitis, obesity, high cholesterol, and type 2 diabetes.

As used throughout, immunotherapy is a therapy that uses the subject’s own immune system to treat cancer in the subject. Examples of cancer immunotherapy include, but are not limited to, monoclonal antibodies, immune checkpoint inhibitors, cancer vaccines, cytokines and interferons. In the methods described herein, the immunotherapeutic agent can be selected from the group consisting of a programmed cell death protein 1 (PD-1) inhibitor, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, a programmed death- ligand 1 inhibitor (PD-L1), a lymphocyte activation gene 3 (LAG-3) inhibitor, a B- and T- lymphocyte attenuator (BTLA) inhibitor, an adenosine A2A (A2aR) inhibitor, or a B-7 family inhibitor. In some methods, the PD-1 inhibitor is an anti-PD-1 antibody. For example, and not to be limiting, the anti-PD-1 inhibitor can be selected from the group consisting of nivolumab, pembrolizumab and pidilizumab. In other methods, the anti-CTLA-4 inhibitor is an anti- CTLA-4 antibody. For example, and not to be limiting, the CTLA-4 inhibitor can be ipilimumab or tremelimumab.

Also provided are methods for treating nonalcoholic steatohepatitis (NASH) in a subject. The methods comprise (a) obtaining a first biological sample from the subject prior to administration of proglumide; (b) measuring the level of one or more miRNAs selected from the group consisting of miR-185- 5p, miR-378a-3p, miR708-5p, miR-346-5p, and miR302d-3p in the first sample; (c) administering to the subject an effective amount of proglumide; (d) obtaining a second biological sample from the subject following administration of proglumide; (e) measuring the level of one or more miRNAs selected from the group consisting of miR-185- 5p, miR-378a-3p, miR708-5p, miR-346-5p, and miR302d- 3p in the second sample; (f) comparing the levels of the one or more miRNAs determined in steps (b) and (e); and (g) administering proglumide to the subject subsequent to the comparing step (f) if the level of the one or more miRNAs determined in step (e) is higher than the level of the one or more miRNAs determined in step (b); or (h) modifying the dosage regimen of proglumide or administering a non-proglumide agent to the subject subsequent to the comparing step (f) if the level of the one or more miRNAs determined in step (e) is not significantly changed or is lower than the level of the one or more miRNAs determined in step (b).

Other methods for treating NASH comprise (a) obtaining a first biological sample from the subject prior to treatment; (b) measuring the level of one or more miRNAs selected from the group consisting of miR-103-3p, miR-142-5p, miR126, miR-711, and miR135b-5p in the first sample; (c) administering to the subject an effective amount of proglumide; (d) obtaining a second biological sample from the subject following administration of proglumide; (e) measuring the level of one or more miRNAs selected from the group consisting of miR-103-3p, miR-142-5p, miR126, miR-711, and miR135b-5p, in the second sample; (f) comparing the levels of the one or more mRNAs determined in steps (b) and (e); and (g) administering proglumide to the subject subsequent to the comparing step (f) if the level of the one or more miRNAs determined in step (e) is lower than the level of the one or more miRNAs determined in step (b); or (h) modifying the dosage regimen of proglumide or administering a non-proglumide agent to the subject subsequent to the comparing step (f) if the level of the one or more miRNAs determined in step (e) is not significantly changed or is higher than the level of the one or more miRNAs determined in step (b).

In some methods, modifying the dosing regimen of proglumide comprises increasing the dosage of proglumide. In some methods, modifying the dosing regimen of proglumide comprises administering proglumide and a second therapeutic agent to the subject. In some methods, the second therapeutic agent is metformin, pioglitazone, vitamin E or a statin (for example, lovastatin, atorvastatin, simvastatin, pravastatin, rosuvastatin or fluvastatin)

Also provided are methods for treating hepatocellular carcinoma in a subject. The methods comprise (a) obtaining a first biological sample from the subject prior to treatment; (b) measuring the level of one or more miRNAs selected from the group consisting of miR- 21a-5p, miR-27a-3p, miR30c-2-3p, and miR-9-5p and miR29b-3p, in the first sample; (c) administering to the subject an effective amount of proglumide; (d) obtaining a second biological sample from the subject following treatment; (e) measuring the level of one or more miRNAs selected from the group consisting of miR-21a-5p, miR-27a-3p, miR30-2-3p, and miR-9-5p and miR29b-3p, in the second sample; (f) comparing the levels of the one or more mRNAs determined in steps (b) and (e); and (g) administering proglumide to the subject subsequent to the comparing step (f) if the level of the one or more miRNAs determined in step (e) is lower than the level of the one or more miRNAs determined in step (b); or (h) modifying the dosage regimen of proglumide or administering a non-proglumide agent to the subject subsequent to the comparing step (f) if the level of the one or more miRNAs determined in step (e) is not significantly changed or is higher than the level of the one or more miRNAs determined in step (b).

In some methods, modifying the dosage regimen of proglumide comprises increasing the dosage of proglumide. In some methods, altering the dosage regimen of proglumide comprises administering proglumide and a second therapeutic agent to the subject. In some methods, the second therapeutic agent is an immune checkpoint inhibitor or a chemotherapeutic agent, as described above.

As used herein, the term therapeutically effective amount or effective amount is defined as any amount necessary to produce a desired physiologic response, for example, treating a disease or disorder. A suitable dose of a therapeutic agent described herein, which dose is capable of treating liver disease in a subject, can depend on a variety of factors including whether it is used concomitantly with other therapeutic agents. Other factors can include medical issues or disorders concurrently or previously affecting the subject (for example, diabetes, high cholesterol, hypothyroidism, etc.), the general health of the subject, the genetic disposition of the subject, diet, time of administration, rate of excretion, drug combination, age or size of the subject, and any other additional therapeutics that are administered to the subject. It should also be understood that a specific dosage and treatment regimen for any particular subject also depends upon the judgment of the treating medical practitioner. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.

Exemplary dosage amounts for administration of a CCK receptor antagonist include doses from about 0.5 to about 200 mg/kg of body weight of active compound per day, which may be administered in a single dose or in the form of individual divided doses, such as from 1 to 4 times per day. Alternatively, the dosage amount can be from about 0.5 to about 150 mg/kg of body weight of active compound per day, about 0.5 to 100 mg/kg of body weight of active compound per day, about 0.5 to about 75 mg/kg of body weight of active compound per day, about 0.5 to about 50 mg/kg of body weight of active compound per day, about 0.5 to about 25 mg/kg of body weight of active compound per day, about 1 to about 50 mg/kg of body weight of active compound per day, about 1 to about 40 mg/kg of body weight of active compound per day, about 1 to about 30 mg/kg of body weight of active compound per day, about 1 to about 30 mg/kg of body weight of active compound per day, about 30 mg/kg of body weight of active compound per day about 20 mg/kg of body weight of active compound per day, about 10 mg/kg of body weight of active compound per day, or about 5 mg/kg of body weight of active compound per day.

The dosage amounts for administration of a CCK receptor antagonist include doses from about 1 mg to 2000 mg of active compound per day, which may be administered in a single dose or in the form of individual divided doses, such as from 1 to 4 times per day. Alternatively, the dosage amount can be from about 100 mg to about 300 mg of active compound per day, about 200 mg to about 600 mg of active compound per day, about 200 mg to about 600 mg of active compound per day, about 200 mg to about 900 mg of active compound per day, about 300 mg to about 900 mg of body weight of active compound per day, about 300 mg to about 1200 mg of active compound per day, about 600 mg to about 1200 mg of active compound per day, about 300 mg to about 1600 mg of active compound per day, about 600 mg to about 1600 mg of active compound per day, about 300 mg to about 2000 mg of active compound per day, or about 600 mg to about 2000 mg of active compound per day. In some examples, about 300 mg, about 400 mg or about 500 mg of proglumide is administered 1, 2, 3 or 4 times a day. One of skill in the art would adjust the dosage as described below based on specific characteristics of the inhibitor and the subject receiving it.

Effective amounts and schedules for administering a therapeutic agent can be determined empirically and making such determinations is within the skill in the art. The dosage ranges for administration are those large enough to produce the desired effect in which one or more symptoms of the disease or disorder are affected (e.g., reduced or delayed). The dosage should not be so large as to cause substantial adverse side effects, such as unwanted cross-reactions, unwanted cell death, and the like. Generally, the dosage will vary with the type of inhibitor, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and severity of the particular condition and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosages can vary, and can be administered in one or more dose administrations daily.

Any of the therapeutic agent described herein can be provided in a pharmaceutical composition. These include, for example, a pharmaceutical composition comprising a therapeutically effective amount of one or more CCK-R antagonists, e.g., proglumide and a pharmaceutical carrier.

Depending on the intended mode of administration, the pharmaceutical composition can be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, capsules suppositories, pills, capsules, powders, liquids, or suspensions, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include a therapeutically effective amount of the agent described herein or derivatives thereof in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, or diluents. By pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, which can be administered to an individual along with the selected agent without causing unacceptable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained.

As used herein, the term carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington: The Science and Practice of Pharmacy, 23d edition, Adeboye Adejare, ed., Elsevier (2021).

Examples of physiologically acceptable carriers optionally include buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; saltforming counterions such as sodium; and/or nonionic surfactants such as TWEEN® (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, NJ).

Compositions containing the agent(s) described herein suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be promoted by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, for example, sugars, sodium chloride, and the like may also be included. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Solid dosage forms for oral administration of the compounds described herein or derivatives thereof include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds described herein or derivatives thereof are admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example, paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art. They may contain opacifying agents and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration of the compounds described herein or derivatives thereof include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers, such as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include additional agents, such as wetting, emulsifying, suspending, sweetening, flavoring, or perfuming agents.

The compositions are administered in a number of ways depending on whether local or systemic treatment is desired and on the area to be treated. The compositions are administered via any of several routes of administration, including orally, parenterally, intravenously, intraperitoneally, intramuscularly, subcutaneously, intrarectally, intracavity or transdermally. Pharmaceutical compositions can also be delivered locally to the area in need of treatment (e.g., to the liver), for example by local application (e.g., during surgery) or local injection. Administration can also be achieved by means of an implant. The implant can be of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. The implant can be configured for sustained or periodic release of the composition to the subject. See, e.g., U.S. Patent Application Publication No. 20080241223; U.S. Patent Nos. 5,501,856; 4,863,457; and 3,710,795; and European Patent Nos. EP488401 and EP 430539. The composition can be delivered to the subject by way of an implantable device based on, e.g., diffusive, erodible, or convective systems, e.g., osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmosis systems, vapor pressure pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, erosion-based systems, or electromechanical systems.

Effective doses for any of the administration methods described herein can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, patent applications and publications referred to throughout the disclosure herein are incorporated by reference in their entirety.

As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to “a miRNA” or “the miRNA” may include a plurality of transcripts.

The use of any and all examples or exemplary language (e.g., “such as”) provided herein, is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

The terms “may,” “may be,” “can,” and “can be,” and related terms are intended to convey that the subject matter involved is optional (that is, the subject matter is present in some examples and is not present in other examples), not a reference to a capability of the subject matter or to a probability, unless the context clearly indicates otherwise.

The terms “optional” and “optionally” mean that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present as well as instances where it does not occur or is not present.

The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements. As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

As used herein, the transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP §2111.03. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise-indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a dosage is stated as 100 mg to 1600 mg, it is intended that values such as 200 mg to 400 mg, 100 mg to 500 mg, or 100 mg to 300 mg, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed and a number of modifications that can be made to a number of molecules including in the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.

EXAMPLES

Non-alcoholic steatohepatitis (NASH) is one of the leading causes of liver-related morbidity and mortality in the world and its incidence has sharply risen since the beginning of the 21 ^st century. Unfortunately, NASH is becoming an important etiologic factor in the development of advanced liver disease and hepatocellular carcinoma (HCC).

The CCK receptor antagonist, proglumide, was previously tested in a murine model of diet-induced NASH. Proglumide was used since it was water soluble and could be added to the animals’ drinking water for chronic administration, and also because proglumide is unique among the CCK receptor antagonists in that it is the only antagonist shown to increase bile flow and decrease bile acid concentration in animal models. In this model, proglumide not only prevented the histologic features of NASH, including inflammation, balloon degeneration and fibrosis, but it was also able to reverse established NASH. One of the most striking findings of this preclinical study was that proglumide prevented the development of HCC in mice whether it was administered as a preventive or therapeutic agent for NASH. Of interest, it was shown that CCK-B receptor expression was significantly up-regulated in livers of mice with NASH, and this receptor becomes over-expressed in HCC. Described below is a Phase 1 study conducted to test the safety and toxicity of proglumide in human subjects with NASH and to determine the recommended Phase 2 dose.

Setting and Referral Process

Subjects were recruited from outpatient liver clinics from two centers: Georgetown University Medical Center, an academic institution, and the Washington DC Veterans Affairs Medical Center, a federal institution. The research protocol was approved by the FDA and the Institutional Review Boards at both sites, and the trial was registered on the www.clinicaltrials.gov website. All participants signed and agreed to the informed consent.

Research Participants and Enrollment Criteria

The inclusion criteria comprised male and female subjects’ ages 18 to 85 years of age with radiographic imaging (by ultrasound, MRI, or CT) of fatty liver disease and elevation in serum transaminases (alanine aminotransferase, ALT or aspartate aminotransferase, AST), and one of the following: BMI>30, hyperlipidemia, or evidence of diabetes based on an abnormal HgbAlc. Subjects were also required to have evidence of mild to moderate fibrosis by Transient elastography FibroScan of Fl to F3 (7-14kPa) and a stiffness score < 14 kPa.

The exclusion criteria included those with evidence of active alcohol use/abuse, and those with chronic viral hepatitis B or hepatitis C, autoimmune hepatitis, or drug induced liver disease. Those with evidence of cirrhosis on exam, histologically, imaging, or with a kPa score on FibroScan >14, or a history of HCC were excluded. Laboratory tests that warrant exclusion include: Leukocyte Count <3.5 K/UL; Hemoglobin <9.5 g/dL; Blood Urea Nitrogen >30 mg/dL (hydrated); Creatinine >2.0 mg/dL, ALT/ AST > 5X ULN, ALP>2X ULN. Evidence of abnormal synthetic liver function including abnormal total bilirubin, and abnormal prothrombin time (PT) or increased INR (unless on warfarin) as well as platelet count <150,000 / mm ³ . Those with a history of gall bladder disease without surgical removal were excluded due to the potential risk of the CCK-receptor antagonist decreasing gallbladder contraction. Since proglumide is primarily cleared by the kidney, those with an estimated glomerular filtration rate (eGFR of < 90 mL/min/1.73m ² were excluded. Subjects with type 1 diabetes mellitus, poorly controlled type 2 diabetes, with HgbAlC > 8, or diabetic patients that have not been on stable doses of anti-diabetic medication for at least 90 days prior to the screening visit were excluded. Subjects that were pregnant or breast feeding, those with bariatric surgery, and those with a known preexisting medical or psychiatric condition that could interfere with the subject’s ability to provide informed consent or participate in study conduct, or that may confound the study findings were not included in the study. Subjects on statins were eligible, and statins were continued at the same dose for the duration of the study.

Study Design and Objectives

This investigation was an open-labeled Phase 1 clinical trial to establish the safety, toxicity, and efficacy of a CCK receptor antagonist, proglumide for treating human subjects with NASH. The primary objective of this Phase 1 investigation was to evaluate the safety and determine the recommended Phase 2 dose (RP2D) of proglumide in eligible subjects with NASH. The secondary objective was to assess the effects of proglumide on serum liver transaminases, fibrosis and steatosis scores by the FibroScan in order to assist in the determination of estimated sample size needed for a phase 2 trial. An exploratory objective included evaluating a noninvasive blood biomarker panel for hepatic fibrosis improvement. The study was conducted using three cohorts of subjects with increasing doses of the investigational drug, proglumide. Six subjects were recruited for each of the three cohorts for a total of 18 participants in this study. Each cohort was treated with proglumide for 12 weeks in sequential order as follows: The first cohort received proglumide at 800 mg/day; the 2 ^nd cohort was treated with proglumide 1200 mg/day, and the third cohort received 1600 mg/day of proglumide. After obtaining informed consent, subjects were evaluated at a screening visit where procedures including a complete history and physical exam, inclusion criteria laboratory tests, radiographic imaging with liver and gallbladder ultrasound, and a fasting FibroScan was obtained. All the screening procedures were completed within 28 days before the baseline visit. Eligible subjects were scheduled for a baseline visit in the Clinical Research Unit where weight and BMI were calculated, an interim examination with vital signs was performed, repeat liver profile was obtained to assure persistent elevation of transaminases, a medication and symptom diary was provided and medication was dispensed. A baseline blood sample was obtained for pharmacokinetic (PK) proglumide blood levels and for research analysis of the liver fibrosis biomarker panel. Subjects were provided a schedule with follow-up visits at week-2, week-4, week-8, week-12, and week 16. At each of the visits vital signs and weight were obtained; laboratory tests (including routine chemistries, liver function, CBC, PT, urinalysis and pregnancy urine test (if necessary) were collected for safety analysis; an interim history and physical exam were performed; the adverse events and drug accountability were evaluated; and a new supply of medication was dispensed. At baseline and at weeks 4, 8, and 12 blood was collected for the proglumide PK levels and biomarker research panel. At week-12, an end-of-treatment right upper quadrant ultrasound was performed to rule out the development of cholecystitis or new gallbladder changes, and a fasting FibroScan was obtained to determine liver stiffness by transient elastography in kPa with steatosis evaluation by Controlled Attenuation Parameter (CAP) score. A follow-up safety visit was scheduled 4 weeks after stopping proglumide (week- 16) and subjects had repeat chemistries and complete blood count performed.

After the subjects in each cohort completed the treatment, the results of the subjects’ laboratory tests, adverse events and imaging results were provided in a blinded fashion to the Safety Monitor for evaluation. The Safety Monitor was a Transplant Hepatologist that was not involved in recruiting, evaluating, or seeing any subjects in the study. The Safety Monitor was required to confirm there were no safety issues in each cohort before recruitment into the next dosing cohort was allowed. Intervention

Proglumide is a water soluble CCK receptor antagonist manufactured using GMP criteria by A.M.S.A. S.p.A. - COSMA S.p.A., Milan, Italy. The bulk compound was found to be >99% pure by HPLC and mass spectroscopy. Proglumide was compounded into vegan capsules with each capsule containing 400 mg of the drug by custom Scripts Pharmacy in Lancaster, PA. Subjects in Cohort 1 were administered proglumide using 400 mg by mouth twice daily for a total daily dose of 800 mg. The second cohort was treated with proglumide 400 mg orally three times daily or 1200 mg/ day, and the third cohort was administered proglumide 800 mg by mouth twice daily (two 400 mg capsules twice daily) or 1600 mg/ day. The investigation pharmacist labeled the research medication at each site and dispensed it every four weeks during the treatment period.

Study Assessments

Laboratory blood tests including a hematology panel, a chemistry panel, a liver profile, prothrombin time / INR, urinalysis, and a pregnancy test (as needed) were evaluated at each visit for safety analysis. Parameters were established in the clinical protocol for early stopping rules and halting of the study for certain elevations in hepatic transaminases and significant changes in other laboratory values.

Subject drug compliance and adverse events were recorded in patient diaries and reviewed at each visit. In addition, a side effect questionnaire was performed at each visit that specifically asked if side effects over the past month included the following: loss of appetite, itching, chest pain, shortness of breath, trouble urinating, diarrhea or constipation, and headache or confusion. Adverse events and any protocol deviations were recorded with the level of severity, whether the investigation drug was held or continued, and the likelihood of whether the side effect was related to proglumide. The adverse events were reported to the safety monitor, the respective IRBs, and FDA at the required intervals or during the annual progress report.

Blood samples were collected to measure proglumide blood levels by mass spectroscopy in the Georgetown University Lombardi Core Metabolomics and Proteomics facility. Serum was isolated from the blood samples for the proglumide measurements and for the research blood analysis for the fibrosis biomarker panel and frozen at -80°C until all subjects in the cohort completed week-12. At that time serum samples from each respective cohort were analyzed together for quality control. Proglumide blood levels were analyzed by mass spectrometry (Xevo-TQ-S, Waters Corporation, USA) and the protocol was optimized using the “Intelli Start” feature of MassLynx software (Waters Corporation). Expanded details of the methods for the analysis of proglumide by mass spectrometry are provided in the Supplemental Materials Methods section.

MicroRNAs (miRNAs) are excreted in the serum and are very stable in the blood. In a previous experiment, a 380 miRNA qPCR-based array was analyzed to examine the fibrotic tissues in mice treated with proglumide compared to controls, and it was found that a significant portion of miRNAs that were upregulated by proglumide also inhibit fibrosis in hepatic cell models (e.g. miR-185- 5p, miR-378a-3p, miR708-5p, and miR-346-5p. Since these four miRNAs are the same in humans as mice and are secreted in the peripheral blood, these four miRNAs were measured as potential biomarkers to determine if proglumide was decreasing fibrosis in the proglumide-treated human subjects of this study. Cell free circulating miRNA was obtained by using serum from study subjects. MiRNA analysis was performed as previously described previously (LaConti et al. “Tissue and serum microRNAs in the Kras(G12D) transgenic animal model and in patients with pancreatic cancer,” PLoS One 201 l;6:e20687). In brief, serum samples were mixed at a ratio of 1 : 10 with Qiazol lysis reagent, and the lysate was extracted with CHC13. The aqueous phase was further processed for total RNA using the miRNeasy Mini kit (Qiagen). Serum miRNA was converted to cDNA using miScriptll RT kit (Qiagen) and miRNA expression profiling was performed using miRNA specific primers and miScript SYBRgreen PCR kit on an ABI 7900 HT Real- Time PCR system (Applied Biosystems). Three replicates were performed for each specimen. A dissociation curve analysis of PCR products was carried out to confirm the specificity of amplification, and the data was normalized using miR-16-5p which was found as appropriate endogenous control. The relative differences between the two groups were calculated using a AACT method. Significance between data from two groups were determined using Fisher’s exact test (p<0.05).

Liver ultrasound imaging and FibroScan were performed by a technician blinded to the baseline reports and interpreted by a staff radiologist or hepatologist, respectively, that were not involved in the research study.

Statistical Analysis

Statistical analysis of de-identified data was performed by a statistician with expertise in clinical trials and not involved in conducting this clinical trial. An intent-to-treat analysis was performed, in which the available data from all patients were included in the statistical analysis. Safety analysis and adverse events were analyzed by the Safety Monitor for the primary outcome of this Phase 1 study. The secondary outcome measurement included changes in liver transaminases and mean values for each cohort at week- 12 compared to baseline values. Values for mean baseline fibrosis kPa values and CAP scores for steatosis were compared using Student’s T-test for changes at week 12 from baseline. Nonparametric Spearman's rho analysis was performed to determine two-tailed correlation coefficients for the percent change in serum ALT, AST, fibrosis kPa score, CAP steatosis score, proglumide blood levels, and for each miRNA. Mean proglumide blood levels were evaluated for each cohort over time to confirm compliance and also to determine whether any proglumide blood level correlated with the reporting of adverse events or toxicity.

Results

Patient Characteristics

Of the 18 subjects enrolled in the study, there were 5 females and 13 males. The ages ranged from 32 to 67 years of age with a mean age of 53.7± 2.3 years and with no significant difference in mean ages between the three cohorts. The mean ± SEM age for Cohort 1 was 58.5 ± 2.39 years; Cohort 2 was 55 ± 4.18 years, and Cohort 3 was 47.5 ± 4.43 years. There was no change in body weight from baseline (96.7±5.1 kg) to week 12 (98.9±5.8 kg). Half of the subjects (N=9) were recruited from the outpatient clinics from Georgetown University and the other half (N=9) were recruited from the DC Veterans Affairs Medical Center. Ethnicity or race included 13 (72%) were white non-Hispanic, 2 (11%) were White Hispanic, 2 (11%) were Black, and 1/18 (6%) was South-Asian. Fourteen of the 18 subjects had BMI measurements > 30 upon enrollment; 10 of the 18 had hyperlipidemia, and 2 of the 18 had type 2 diabetes mellitus requiring anti-hyperglycemic medication. All of the subjects had previously been instructed by their primary care providers to diet, exercise and lose weight.

Safety Measurements

Overall, proglumide was well tolerated at all three doses without any serious adverse events. All 18 of the subjects enrolled completed the study and none dropped out due to side effects. Only 9 side effects were reported in 6 of the subjects, and all of these events were reports in subjects from Cohort 1 or Cohort 2. There were no adverse events reported in those receiving the highest dose of proglumide or Cohort 3. Of the side effects reported, gastrointestinal side effects were most frequent including nausea (1), loss of appetite (1), abdominal pain (2), and constipation (3). All of the reported side effects were mild and resolved without discontinuing or holding proglumide. The majority of the adverse events reported occurred within the first month of initiating therapy with proglumide.

Laboratory blood tests and urinalysis were also checked at baseline and weeks 2, 4, 8, 12 and 16. Compared to baseline values, there were no toxicities recorded for standard laboratory tests in subjects from all three cohorts. Gallbladder and liver ultrasound were performed at baseline and after 12 weeks of proglumide since acute cholecystitis has been reported with more potent, CCK-A receptor antagonists. One of the 18 subjects in Cohort 1 had gallstones without cholecystitis at baseline and there was no change in this subject’s gallbladder imaging after 12- weeks of proglumide therapy. Two subjects had prior cholecystectomy. None of the subjects developed cholecystitis or new gallstones during the 12-week course of therapy. All subjects had imaging evidence of steatosis at baseline by liver ultrasound as per the inclusion criteria. By week-12, two of the subjects (from Cohort 3) no longer had radiographic evidence of hepatic steatosis by liver ultrasound; the remainder still had evidence of steatosis.

Effects of proglumide therapy on hepatic transaminases

There were no adverse events reported involving an increase in transaminases in any of the treatment groups during the study. The median percent change in serum ALT was +8.42, -5.05, and -22.23 for cohorts 1, 2, and 3, respectively with significance observed with the highest dose of proglumide (P < 0.05). The median percent change in serum AST was - 16.94, -14.33, and -18.52 for Cohorts 1, 2, and 3, respectively. The baseline serum ALT value for all eighteen subjects was 1.79-times the upper limit of normal with a mean value of 73.4 ± 11.1 units/ L (normal range, 15-41 units/L). The mean baseline serum AST value for all subjects was 48.2 ± 5.1 units/L (normal range, 3-34 units/L). Typically subjects with NASH have ALT levels 1.5 to 4 times the upper limits of normal with the ALT value being higher than the AST in those without cirrhosis. Therefore, the population’s transaminase values were characteristic of those with NASH without advanced fibrosis.

Effects of proglumide on hepatic fibrosis and steatosis by transient elastography

Transient elastography utilizing FibroScan was measured in kPa for an estimation of hepatic fibrosis. Of the 18 subjects enrolled, FibroScan at baseline included N=12 with Fl fibrosis, N=3 with F2 fibrosis and N=3 with F3 fibrosis. Cohort 1 subjects included fibrosis scores of Fl (N=4), F2 (N=l), and F3 (N=l). Cohort 2 included fibrosis scores of Fl (N=5) and F2 (N=l); and cohort 3 included those with fibrosis scores of Fl (N=3), F2 (N=l), and F3 (N=2). The median percent change from baseline values to week-12 of the study for liver stiffness measured in kPa using FibroScan was +8.13, -5.44, and -28.87, respectively for cohorts 1, 2, and 3. Compared to the lower dose of proglumide (800 mg/day), change in fibrosis scores of those on the highest dose of proglumide (1600 mg/day) nearly reached significance ( =0.07) even with this small sample size.

At baseline steatosis measured by CAP scores in dB/m with FibroScan included mild steatosis SI (CAP 238-259) (N=l), moderate steatosis S2 (CAP 259-292) (N=5), and severe steatosis S3(CAP>292) (N=12). Steatosis measurements by CAP scores improved in all three cohorts with proglumide over the duration of the study (P= 0.047). The median percent change in CAP scores from baseline values to week-12 was -1.27, -13.27, and -3.38, for cohorts 1, 2, and 3, respectively.

Proglumide blood levels by mass spectrometry

All of the subjects’ baseline blood proglumide levels were undetectable. As expected, those taking the highest dose of proglumide (1600 mg/day) had the highest blood levels measured by mass spectrometry. Proglumide blood levels at week-12 are plotted for each cohort (FIG. 1 A) showing that the mean levels increased with each ascending dose administered. The mean proglumide blood levels measured at baseline, and weeks 4, 8, and 12 are shown for each treatment cohort (FIG. IB). The peak serum concentration or (Cmax) was reached in all of the subjects from cohort 1, and five out of six of the subjects in cohort 2 by week 8. In cohort 3, half of the subjects had higher proglumide blood levels detected at week 12 than at week 8, suggesting that cohort 3 had not yet reached steady state. All three subjects with stage F3 fibrosis by transient elastography had Cmax proglumide values exceeding 24,000 ng/ml. Two of these subjects were in cohort 3 with proglumide Cmax values of 25,065.9 and 24,216. Ing/ml, respectively; however, neither of these subjects experienced side effects or adverse events. The third subject with F3 fibrosis from cohort 1 had a proglumide Cmax value at week 8 of 26,219.5ng/ml. This subject (Subject 2) reported side effects with proglumide including nausea, constipation, and increased thirst. It is uncertain whether the side effects reported by this individual were related to the higher proglumide values, because others reported GI side effects without elevated proglumide blood levels. These results may however suggest that those with more advanced liver fibrosis have the potential for acquiring higher blood levels; and if side effects are reported in these subjects, dose reduction may be warranted. Measurement of serum microRNAs as assessment of liver fibrinolytic activity

Four miRNAs that inhibit hepatic stellate cell activation and liver fibrosis were measured at baseline and during the course of the study. Serum levels of all four of the miRNAs measured, including miR-185- 5p, miR-378a-3p, miR708-5p, and miR-346-5p, increased with proglumide therapy compared to baseline values (FIG. 2). Significant differences compared to baseline measurements were found with miRNA-378-3p (P=0.004), miRNA-708-5p (P<0.001), and miRNA-346-5p (P<0.001). MiRNA values appear to decrease with higher doses of proglumide and likely due to competition of the drug with the miRNA binding proteins such as albumin or Argonaute2.

This study represents a novel approach to managing the NASH epidemic by targeting the CCK receptor signaling pathway. Furthermore, the trial was based upon evidence from murine models of NASH and represents true bench-to- bedside translational research. This investigation met its primary outcome by demonstrating that oral proglumide had a broad safety profile, and was well tolerated without toxicity in subjects with F1-F3 fibrosis and clinical NASH. The few side effects reported were minor and none led to discontinuation of the drug. Subjects were compliant with self-administration according to patient diaries and also confirmed by measurement of proglumide blood levels. Although Phase 1 studies are not designed to determine efficacy, important changes in hepatic transaminases, fibrosis, and steatosis score, that would predict proglumide to be beneficial in proceeding with a Phase 2 clinical trial, were observed. Even though this sample size was small, a significant improvement was noted over the 12-week period in ALT and steatosis scores and near significant decline in fibrosis scores as determined by FibroScan. These changes occurred without any confounding documented weight loss; in fact the mean body weight of the subjects slightly increased over the study in spite of the teams’ recommendation to encourage healthy diets, weight loss, and exercise. Therefore, the improvements in hepatic transaminases and FibroScan scores most likely were the result of taking proglumide.

Three doses of proglumide were used in this study; a low, medium, and high dose. Proglumide was developed years ago for the treatment of peptic ulcer disease; however, commercialization was halted with the development of the more potent proton pump inhibitors. These early trials included over 600 subjects in 15 clinical trials that confirmed the broad safety profile of this compound. Pharmacokinetic studies performed in rats and human subjects showed the metabolism and kinetics in the rats and humans were comparable and that the recommended oral dose of proglumide in man was 400 mg TID or 1200mg/ day. A PK single dose study testing proglumide in subjects with Child- Pugh A and B cirrhosis with normal renal function was performed and it was found that the Cmax and Tmax were comparable to that of healthy controls. Since the current Phase 1 study was the first clinical trial in subjects with NASH, a proglumide dose that was lower (800 mg/day) and a dose higher (1600 mg/day) than the 1200 mg/ day dosing used in earlier peptic ulcer disease studies of healthy subjects without liver disease was selected. Although all three doses were well tolerated in subjects with NASH, the greatest decline was observed in ALT and liver stiffness by FibroScan using the highest dose (1600 mg/ day). Furthermore, there were no reported adverse events in subjects administered the highest dose of proglumide; therefore, 1600 mg/day is a suitable does to move forward into a Phase 2 clinical trial. Measurement of proglumide blood levels was beneficial in this study because they not only confirmed patient compliance and a dose-related effect, but it was found that three subjects with advanced F3 fibrosis experienced the highest Cmax proglumide values. With longer duration of therapy, as will be needed in a Phase 2 trial, a protocol was developed that can accurately measure proglumide blood levels using mass spectrometry in the event that side effects occur, thus allowing for measurement and dose reduction if necessary.

Although liver biopsies are generally safe in those with mild liver disease, they are not without risk, and also deter some subjects from participation in research trials. Investigators have been studying noninvasive tests to measure hepatic fibrosis ranging from simple biomarker blood tests such as the FIB-4 and APRI tests, and more complex biomarker panels that include proteins involved in fibrinolysis or fibrogenesis such as TEMPI and hyaluronic acid. In mouse models, it was found that a significant portion of miRNAs that were upregulated by proglumide in tissues and blood (e.g., miR-185-5p, miR-378a-3p, miR708-5p, miR-346-5p) had previously been shown to inhibit fibrosis, inflammation, or both in activated hepatic stellate cells. Similarly, miRNAs that were down-regulated by proglumide are clearly shown to promote fibrosis, inflammation or both in hepatic stellate cells (e.g.miR-21, miR-103-3p, miR-142-5p, and miR-126). Since an elevation in miRNAs in the peripheral blood is more reliable for measurement by PCR than a decrease, the first four miRNAs were selected to test in participants’ blood during this Phase 1 trial. A significant fold-increase in the expression of these miRNAs could indicate there was fibrinolysis or cessation of fibrogenesis in the livers of study subjects while administered proglumide.

In conclusion, therapy with the oral CCK receptor antagonist, proglumide, is safe and well tolerated in subjects with clinical NASH. Analysis of miRNAs

Specific microRNAs (miRNA) that epigenetically regulate fibrosis genes were changed by proglumide (Heat map, FIG. 3). As set forth above, in murine models, many of these microRNAs are stable and easily detected in the blood. Most importantly, the microRNAs selected for analysis are the same in humans as in mice and serve the same function. Since miRNAs are stable in the peripheral blood a panel of miRNA biomarkers can be used as a noninvasive test. Those miRNAs upregulated by proglumide in the pancreas have been shown to inhibit fibrosis, inflammation or both including miR-185-5p, miR-346- 5p, miR-378a-3p, and miR708-5p. In addition to using a miRNA biomarker panel, the subjects’ peripheral blood can also be analyzed for hyaluronan and propeptide of type III collagen.

MicroRNA biomarker blood panel corrects with changes in TME and invasiveness ofPDAC It was demonstrated in two murine models ofPDAC, that proglumide decreases fibrosis and alters the immune signature of the pancreatic TME rendering the tumor sensitive to gemcitabine chemotherapy. Blood samples were collected from mice to evaluate if the microRNAs altered by proglumide in the heat map shown in FIG. 3 could be detected in the blood and correlated with the histologic changes identified in a tumor microenvironment (TME) in regards to fibrosis in the TME and invasiveness. When examining the blood levels of microRNAs between mice treated with gemcitabine monotherapy compared to those treated with the combination of gemcitabine and proglumide, significant differences were found that correlated histologically with changes identified in the TME. The microRNAs (miR) miR-185-5p, miR-346-5p, miR-378-3p, miR-708-5p inhibit fibrosis from stellate cells and cancer associated fibroblasts. These microRNAs were significantly upregulated in the peripheral blood of mice treated with combination therapy (i.e., proglumide and gemcitabine), as shown in FIG. 4. miR-141, miR205, and miR-200b are members of the miR- 200 family. These three markers were significantly upregulated in combination treated mice compared to the gemcitabine monotherapy mice in the PANC-1 immune deficient PDAC model (FIG. 5).

These studies demonstrated that proglumide alters fibrosis and immune cells in the TME rendering the tumor susceptible to gemcitabine therapy, and these changes are epigenetically regulated by microRNAs. Since microRNAs are excreted from tumors as exosomes and are very stable in the blood, microRNAs can be developed as biomarkers of liver disease. As shown herein, microRNAs associated with tumor invasion- metastasis and fibrosis, that are changed by proglumide therapy, can be detected in the mouse blood and their levels correlate with the histologic changes and metastatic potential of the primary tumors. miRNA biomarker panel for fibrosis and invasion

Using blood from preclinical murine PDAC studies, where mice were treated with gemcitabine monotherapy or the combination of gemcitabine and proglumide, murine blood was collected at the time of necropsy and RT-PCR was performed for some of the selective miRNAs found in the Heat map (FIG. 3) associated with fibrosis and inflammation of the TME that were indeed upregulated by proglumide therapy. Blood from mice in each group was available for miRNA analysis (FIGS. 4 and 5). These data show that miRNAs can be measured in the peripheral blood and these changes in miRNA levels could be a sensitive method to monitor proglumide response without a biopsy. miRNAs from the heat map that are associated with decreasing fibrosis and oncogenesis of the TME were selected. The selected miRNAs were the same and have the same functions in mouse and humans (Table 2). Since the panel of selected miRNAs mostly increase in the blood with less fibrosis and less invasion potential, the feasibility of measurement should be reliable and reproducible in the blood from human subjects.

Table 2. Mouse and human miRNAs related to fibrosis and oncogenesis that changed in pancreatic tissue with proglumide is also detected in the mouse blood.

In brief, 400 pl serum samples are mixed at a ratio of 1 : 10 with Qiazol lysis reagent.

The lysate will be extracted with CHCh and the aqueous phase will be further processed for total RNA using the miRNeasy Mini kit (Qiagen, Hilden, Germany). miRNA will then be converted to cDNA using miScript-II RT kit. miRNA expression profiling for specific miRNAs will be performed using mi Script primer assays and SYBRgreen PCR kit on an ABI 7900 HT Real-Time PCR system (Applied Biosystems, Waltham, MA). Three replicates will be performed for each specimen. A dissociation curve analysis of PCR products will be carried out to confirm the specificity of amplification. Data will be normalized using appropriate endogenous controls. Changes in expression will be calculated using the mean ACT method; values for each group will be compared using a modified Bonferroni method to correct for multiple comparisons. Principal component analysis and hierarchical clustering will be performed based on the mean centered and scaled miRNA expression levels. The clustering methods will use XLSTAT (Addinsoft Inc.) within Excel on OSX 10.7.5. These methods allow for the calculation of significance between the hierarchical clusters and derive p-values using Fisher’s exact test. Receiver operating characteristic (ROC) analysis will be used to assess the performance of the classifier model. Using R statistical package the area under curve (AUC) will be calculated and analyzed. Due to the potential for a large number of candidate biomarkers (N=15) to be examined here, a regularized logistic regression approach will be used for feature selection. A 10-fold cross-validation approach will be used to determine tuning parameters and calibrate the prediction model in the discovery set. Whether this miRNA panel can be evaluated to predict progression in this cohort will be examined. To do this, the hazard ratios will be determined by building an iterative Cox model wherein mean values of all 15 miRNAs will be used as categorical predictors.

Methods hyaluronan and propeptide of type III collagen (PRO-C3)

In order to validate a miRNA biomarker panel and show the changes that are found in fibrosis miRNAs associated with the TME, results will be compared with the miRNAs in subjects’ serum to that of hydroxyproline, serum hyaluronan (HA), and propeptide of type III collagen using assays that have been used in human subjects with PDAC. Levels of hydroxyproline will be determined using mass spectrometry as previously described (Jolly et al., Cholecystokinin receptor antagonist induces pancreatic stellate cell plasticity rendering the tumor microenvironment less oncogenic, Cancers 15:2811 (2023)). Pretreatment and longitudinal serum concentrations of HA will be determined by a human HA high-sensitive solid-phase immunoassay (Quantikine® Immunoassay, R&D Systems) as previously described in subjects with PDAC (Chen et al. “Clinical value of serum hyaluranon and propeptide of type III collagen in patients with pancreatic cancer,” Int. J. Cancer 146: 2913- 22 (2020)). The lower level of detection in this assay for HA is 0.068 ng/ml. The inter-assay coefficient of variation (CV) is <7.2% and the intra-assay CV is <4.9%. The molecular weight range of HA detected by the Quanitkine kit is 0.2-40.0 ng/ml. Serum PRO-C3 will be determined by enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody to detect the N-protease mediated cleavage of the N-terminal propeptide of type III collagen (Nordic Bioscience, PRO-C3 assay, Herlev, Denmark) (Nielsen et al. “The neo-epitope specific PRO-C3 ELISA measures true formation of type III collagen associated with liver and muscle parameters,” Am. J. Transl. Res. 5: 303-315 (2013)).

Statistical Analysis

Mean baseline miRNA values will be compared to the mean miRNA values obtained at week-8 using the paired T-test. In addition, the Repeated Measure ANOVA will be used to examine longitudinal changes in measures from baseline and every 8 weeks and will be compared between those receiving gemcitabine & placebo to those treated with gemcitabine & proglumide. Receiver operating characteristic (ROC) analysis will be conducted to assess the diagnostic values of serum miRNAs, HA, and PRO-C3. Changes of serum miRNA panel, HA and PRO-C3 at baseline and after systemic treatment will be investigated. Hazard ratios (HRs) adjusted for chemotherapy regimen (gemcitabine with nab-paclitaxel or with proglumide) will be estimated with Cox proportional-hazard regression for the time-to- progression outcome.

The biomarker panels described herein can be used clinical trials using proglumide or any other compound(s) being investigated to treat hepatic disease, for example liver cancer, and perhaps not have to subject the patients to biopsies. Exosomes act as carriers of miRNAs and thereby protect them from degradation; thus miRNAs are very stable in the blood. Also because there is strong evidence from the preclinical studies described herein that these particular miRNAs change with proglumide treatment and are directly correlated to fibrosis of the TME, this panel will be useful to facilitate response in future clinical trials. All the miRNAs in the panel including the normalizer (U6) had robust Ct value data in serum analysis, and thus appear to be highly suitable as potential biomarkers in the prediction of response. However, the panel(s) can be refined by including additional miRNAs from FIG. 3 which have also been shown to target proteins implicated in fibrosis. In some instances, a miRNA panel described herein can be compared with established TME biomarkers for additional validation of the panel.